Background: There is a low threshold of pain in newborns, especially premature infants, who are extremely sensitive to pain and painful procedures by showing a strong response. This study aimed to determine the effect of facilitated tucking with simulated hands on physiological factors of pain during venipuncture in premature infants. Methods: This experiment was conducted on preterm infants admitted to the neonatal intensive care unit of Amin and Shahid Beheshti hospitals in Isfahan, Iran in 2016. In total, 66 patients were selected through convenience sampling and were randomly assigned to the intervention (N=33) and control (N=33) groups. In the intervention group, venipuncture procedure was performed as infants were placed in the facilitated tucking position using simulated hands. Data were collected applying the new Iranian S1800 monitors for hemodynamic control. Data analysis was performed in SPSS version 18 using Kolmogorov-Smirnov, Chi-square, independent t-test and repeated measures ANOVA. Results: In this research, a statistically significant difference was observed between the mean of arterial oxygen saturation and respiratory rate of the intervention and control groups (P